L-({60 s,5s)-{60 -amino-3-chloro-2-isoxazoline-5-acetic acid

ABSTRACT

Antibiotic AT-125, producible by culturing Streptomyces sviceus in an aqueous nutrient medium has the following structure: The absolute configuration of AT-125 was determined to be L-( Alpha S,5S)- Alpha -amino-3-chloro-2-isoxazoline-5-acetic acid. It is an amphoteric compound and can exist in different ionic forms according to the pH of the environment. At low pH, AT-125 exists in the acid-addition salt form, at a higher pH in a zwitterion form, and at a still higher pH in a metal salt form. AT-125 inhibits the growth of Bacillus subtilis, Saccharomyces pastorianus, Penicillium oxalicum, Candida albicans, Saccharomyces cerevisiae and Escherichia coli, and can be used to inhibit such microorganisms in various environments.

nit

States Patent [191 lilanlta et al.

[ lL-(aS,5S)-0z-AMINO-3-CHLORO-2- IISOXAZOLINE-S-ACETIC ACID [75] Inventors: Ladislav J. llanka; David G. Martin,

both of Kalamazoo, Mich.

[73] Assignee: The Upjohn Company, Kalamazoo,

Mich.

[22] Filed: Feb. 2, 1973 [21] Appl. No.: 329,001

Related US. Application Data [63] Continuation-impart of Ser. No. 234,347, March 13,

1972, abandoned.

[52] US. Cl. 260/307 F, 199/36 R, 424/272 [51] lint. Cl C07d 85/16 [58] Field of Search 260/307 F [56] References Cited OTHER PUBLICATIONS lwasakiC.A. 72, 43647 v. (1970).

Primary Examiner-Raymond V. Rush Attorney, Agent, or Firm-Roman Saliwanchik ABSTRACT Antibiotic AT-l25, producible by culturing Streptomy- [4 1 cc. 24, W74

ces sviceus in an aqueous nutrient medium has the following structure:

form, and at a still higher pH in a metal salt form.

AT-125Hhhibits the growth of menus subtilis,

Saccharomyces pastorianus, Penicillium oxalicum, Candida albicans, Saccharomyces cerevisiae and Escherichia coli, and can be used to inhibit such microorganisms in various environments.

5 Claims, N0 Drawings CROSS REFERENCE TO RELATED APPLICATION This application is a continuation-in-part of pending application Ser. No. 234,347, filed on Mar. 13, 1972, and now abandoned.

BRIEF SUMMARY OF THE INVENTION AT-125 is an amphoteric antibiotic which is producible by culturing an AT-125-producing actinomycete in an aqueous nutrient medium. This antibiotic has the property of adversely affecting the growth of Gram positive and Gram-negative bacteria, for example, Bacillus subtilis and Escherichia coli. It is also active against various fungi and yeast, for example, Saccharomyces pastorianus, Penicillium oxalicum, Candida albicans, and Saccharamyces cerevisiae. Accordingly, the novel antibiotic of the subject invention can be used alone or in combination with other antimicrobial agents to prevent the growth of, or reduce the number of bacteria, yeast, and fungi, as disclosed above, in various environments. For example, it can be used for treating breeding places of silkworms, to prevent or minimize infections caused by Bacillus subtilis.

DETAILED DESCRIPTION Characterization of AT-125 The structure of AT-l 25, consistent with determined spectral and analytical data, is

and its ion and zwitterion forms.

The NMR spectrum of a saturated solution of AT- 125 in D was determined on a Varian XL-IOO spectrometer. The 100 MH spectrum was not first order and clearly indicated the presence of 4 nonexchangeable protons. The methylene protons on C were seen as a typical deceptive 5 line AB pattern with the strongest signal at 350 H and an apparent doublet at 359 H and 361 H The neighboring proton on C was seen as an apparent doublet of triplets centered at 5.36 8 with line separations of 3.5 H and 10 H The signal for the amino acid methine proton was a first order doublet at 4.128 with J 3.5 H The IR spectrum ofa Nujol mull was determined on a Perkin Elmer 421 spectrophotometer. Major peaks (except Nujol) were found at 3030 (broad), 2730. 2620, 1620, 1590, 1495, 1425, 1405, 1325, 1307, .1293, 1275, 1145, 1127, 895, and 870 cm.

The chemical structure of AT-125 in the crystalline form was established rigorously by X-ray diffraction. Crystalline AT-125 can be named chemically as L- (aS,5S)a-amino-3-chloro-2-isoxazoline-5-acetic acid.

Molecular Weight Rigorous X-ray structure established AT-125 as C H,ClN O or molecular weight as 178.6.

Solubilities Soluble in H 0 and slightly soluble in methanol. Relatively insoluble in ethyl acetate. ether, benzene and chloroform.

Antimicrobial Properties of AT-l 25 The following results were obtained with a standard disc plate assay using 13 mm. paper discs and a concentration of 1 mg./ml. of AT-125.

Zone of Inhibition (mm) around a Microorganism 13 mm. Paper Disc Bacillus .rnhlilis (in synthetic agar) 78 Bacillus sub/ills (in nutrient agar) 0 Laclobucillu: cuxei 0 Sarcina lutcu 0 Stapliylmuccns aurcus Mycobacterinm avium 0 Escherichia coli (in nutrient agar) 0 Escherichia coli (in synthetic agar) 36 hazy Salmonella sclmrrmuelleri 0 'Proleux vulguris 0 Klehsicllu pneunwniae 0 Sacc/ruromyces paslvrlanus 53 Penicillinm uxulicum 32 Candida alhicans (tested at ag/ml.) 20 Saccharomyces cererixiae tested at 100 Lg/ml.) 38

THE MICROORGANISM DESCRIPTION Streptomyces sviceus Dietz sp. n.

Color characteristics Aerial growth gray. Melanin positive. The color pattern on Ektachrome [Dietz, A. 1954. Ektachrome transparencies as aids inactinomycete classification. Ann. NY. Acad. Sci. 60:152-154] is given in Table 1. Reference color characteristics are given in Table 2. The culture may be placed in the Gray (GY) and White (W) color series of Tresner and Backus [Tresner, H. D., and E. J. Backus. 1962. System of color wheels for streptomycete taxonomy. Appl. Microbiol. 11:335-338].

Microscopic characteristics Oval to rectangular spores sparsely adorned with warts or short spines borne on candelabra-like sporophores. Sporophores are long, straight and coiled at the ends. Sporophores may be placed in the spiral (S) group of Pridham et al. [Pridham, T. G., C. W. Hesseltine, and R. G. Benedict. 1958. A guide for the classification of Streptomyces according to selected groups. Placement of strains in morphological sections. Appl.

Microbiol. 6:52-79]. Spore surfaces may be placed in the warty group of Dietz and Mathews [Dietz, A., and John Mathews, 1970. Classification of Streptomyces spore surfaces into five groups. Appl. Microbiol. 2l:527533].

Cultural and biochemical characteristics; See Table 3. Carbon utilization The growth of the culture on carbon compounds was determined using the procedures of Pridham and Gottlieb [Pridham, T. G., and D. Gottlieb. 1948. The utilization of carbon compounds by some Actinomycetales as an aid for species determination. J. Bacteriol. 562107-114] and Shirling and Gottlieb [Shirling, E. S., and D. Gottlieb. I966. Methods for characterization of Streptomyces species. Int. J. Sys. Bacteriol. 16:313-340]. In the procedure of Pridham and Gottlieb, S. sviceus showed fair growth on the basal medium, dulcitol, D-sorbitol, salicin, and sodium oxalate; good growth on D-xylose, L-arabinose, rhamnose, D- fructose, D-galactose, D-glucose, D-mannose, maltose, sucrose, lactose, cellobiose, raffinose, dextrin, inulin, soluble starch, glycerol, D-mannitol, inositol, sodium acetate, sodium citrate, and sodium succinate; poor growth on phenol, sodium formate, and sodium tartrate; no growth on cresol and sodium salicylate. In the procedure of Shirling and Gottlieb the culture did not grow on the negative control (basal medium only). Growth was good on the positive control (basal medium plus glucose). Growth was equal to or greater than the glucose control on the basal medium plus L- arabinose, sucrose, D-xylose, inositol, D-mannitol, D- fructose, rhamnose, and raffinose, growth was doubtful with cellulose.

Temperature The culture grew well at l8C.-28C. on Bennett's and Czapeks sucrose agars; at 18C-3 7C. on maltosetryptone agar. There was no growth at 45C. and 55C. on Bennetts, Czapeks sucrose, and maltose-tryptone agars.

Antibiotic-producing properties The culture produces the antimetabolite antibiotic AT-l25.

Source Soil DISCUSSION Streptomyces sviceus is an actinomycete with characteristics of the genus Streptomyces as set forth in Bergeys Manual of Determinative Bacteriology, 7th edition lBreed, Robert S., E. G. D. Murray, and Nathan R. Smith. I957. Bergey's manual of determinative bacteriology, 7th Edition, the Williams and Wilkins Co., Baltimore]. The culture, which was isolated from soil, is distinctly different from Streptomyces species in the Upjohn culture collection and, insofar as can be determined, from those in the literature descriptions in Bergeys Manual, supra, Gauze [Gauze, G. F., T. P. Preobrazenskaya, E. S. Kudrina, N. O. Blinov, I. D. Ryabova, and M. A. Iveshnikova. I957. Problems in the classification of antagonistic actinomycetes. State Publishing House for Medical Literature, Moscow. English edition translated by Fritz Danga; David Gottlieb (ed.). The American Institute of Biological Sciences,

Washington, DC], Hutter IHutter, R. I967. Systematik der Streptomyceten unter besondere Berucksictigung der von ihnen gerbildeten Antibiotica. S. Karger, Basel], Krassilinkov [Krassilnikov, N. A. I949. Actinomycetes. In Guide to the identification of bacteria and Actinomycetes. Academy of Sciences, U.S.S.R., Moscow. English edition translated by J. B. Routien, Chas, Pfizer and Co., Inc., 1957], Waksman [Waksman, S. A. 196 l. The actinomycetes. Vol. 2, Classification, identification, and descriptions of genera and species. The Williams and Wilkins Co., Baltimore], and Shirling and Gottlieb lShirling, E. B., and D. Gottlieb. 1968. Cooperative description of type cultures of Streptomyces. II. Species descriptions from first study. Int. J. Sys. Bacteriol. 18:69-189; Cooperative description of type cultures of Streptomyces. III (1968) Additional species descriptions from first and second studies. Int. J. Sys. Bacteriol. l8:279-392; Cooperative description of type cultures of Streptomyces. IV. (1969) Species descriptions from the second, third and fourth studies. Int. J. Sys. Bacteriol. 19:391-512].

Streptomyces sviceus showed some similarity to Streptomyces liawaiiensis [Cron, M. J., D. F. Whitehead, I. R. Hooper, B. Heinemann, and J. Lein. 1956. Bryamycin, a new antibiotic. Antibiotics and Chemotherapy 6:63-67] ATCC l22236. Both cultures are melaninpositive and have similar carbon utilization patterns in the synthetic medium of Pridham and Gottlieb. Streptomyces lrawaiiensis has open spiral sporophores which are shorter and less distinctive than those of the new culture which are long and coiled at the tip. The spores, as observed by transmission electron microscopy, are round to oval and covered with fine spines for S. lzawaiiensis and oval to rectangular with a sparsely warty to spiny surface for S. sviceus. Distinguishing characteristics are cited in Table 4.

S. sviceus is easily distinguished by its distinctive color pattern and microscopic characteristics from charaterized species of Streptomyces in the Upjohn culture collection and, as far as can be determined, from those cultures characterized in the literature. The cultural characteristics cited in the tables reinforce the distinctive features of S. sviceus. A unique property of this organism in its ability to produce antibiotic AT- I25. It is proposed that the organism characterized herein be considered a new species of Streptomyces designated Streptomyces sviceus (from svice or svicen Czech for candle or candle holder sporophores of cultures are candelabra-like) Dietz, sp. n. the type species is designated the type variety Streptomyces sviceus var. sviceus in accordance with Rule 911(2) of the International Code of Nomenclature of Bacteria [International Code of Nomenclature of Bacteria. 1966. Edited by the Editorial Board of the Judicial Commission of the International Committee on Nomenclature of Bacteria. Intern. J. System. Bacteriol. 16:459-490].

TABLE I Color Pattern of Slreplomyces sviceus on Ektachrome Dielz, A., Ektachronie Transparencies as Aids in Aclinomycete Classification".

Annals of the New York Academy of Sciences. 6(l:I5Z-l$4. I954.

Reference Color Characteristics of Strepwmycer .rt'it'eus Color Harmony Manual,

lSCC-NBS Method of Designating Color and a Dictionary of Color All readings were made using the glossy surface of the chips.

Jacobson. l-Ir. W. C. Granville. and (C l ('hicago. Illinois.

" Kelly. K. l... and I). I3. Judd. I955. The ISCUNBS Method of Designating (olurs and a Dictionary otColur Nantes. US Dept.

Commerce Ciro. 553.

TABLE 3 Agar Medium 3rd ed.. 1948* Names. Circular 553. l955** Bennett's S 3fe silver gray 63 gm light brownish gray R 3ni clove brown 77 to moderate yellowish brown 95g moderate olive brown P 3ig beige brown. mist 80m grayish yellowish brown brown Czapeks S 3lc amber. butterscotch 7l m moderate orange yellow sucrose R Zge covert tan. griege 94 m light olive brown 109 gm light grayish yellow P 3ge beige. camel 79m light yellowish brown Maltose I S a white 263 gm white tryptone R 3 lg adobe brown. cinnamon 77 gm moderate yellowish brown brown. light brown P 3ig camel. maple sugar. tan 80m grayish yellowish brown 95 g moderate olive brown Yeast extract- S 3fe silver gray 63 gm light brownish gray malt extract (lSPQ) R 4nl chocolate. dark brown 64 m brownish gray 81 g dark grayish yellowish brown P 3li beaver 80m grayish yellowish brown 95 g moderate olive brown Oatmeal S Ste silver gray 63 gm light brownish gray (lSP-3l R Zig slate tan to llOg grayish yellow 3ml beaver gray l 12m light olive gray to 96g dark olive brown 266m dark gray P Zihdark covert gray l 12m light olive gray 265m medium gray lnorganic- S 3t'e silver gray 63 gm light brownish gray salts starch llSP4) R 3ge beige. camel 79m light grayish yellowish brown 94m light olive brown P 3ge beige. camel 79 m light grayish yellowish brown 94m light olive brown Glycerol S C light gray 264 gm light gray asparagine (lSP-S) R 3ml beaver gray 96g dark olive brown P 3fe silver gray 63 gm light brownish gray S Surface R Reverse l Pigment Foss. 194K. (olor Harmony Manual. 3rd Edition. Container Corporation of America.

Cultural and Biochemical Characteristics of Slreprumyvox .rvit-eus Medium Surface (aerial growth) Reverse Other Characteristics Agar Media Peptone-iron Trace gray Brown Brown pigment (melanin (aleium malate Glucose asparagine Skim milk Tyrosine Poor gray-white Poor lavender-gray Very slight trace gray Poor gray White to cream Olive Yellow-tan-brown Tan brown positive) Yellow-tan-brown pigment Casein not solubilized Brown pigment Tyrosine solubilized TABLE 3-Continued Medium Surface (aerial growth) Reverse Other Characteristics Xanthinc Nutrient starch Poor gray-white Fair gray'white Cream-tan Cream-yellow Cream-tan pigment Xanthine solubilized Yellow pigment Starch hydrolyzed Yeast Extract- Good gray Brown Tan-brown pigment malt extract Peptone-yeast- Brown Brown pigment (melaninextract-iron positive) (lSP-(al Tyrosine Gray Brown Brown pigment (melanin- (lSP-7) positive) 507:

No pigment (melaninnegative) 5071 Gelatin Media Plain Trace white on surface Yellow to olive pigment pellicle No liquefaction Nutrient Olive tan pigment No liquefaction Broth Media Synthetic nitrate Colorless surface Pale yellow pigment pellicle Compact bottom growth Nitrate not reduced to nitrite Nutrient nitrate Colorless surface Brown pigment pellicle Compact bottom growth Nitrate not reduced to nitrite Litmus milk Brown surface ring Litmus reduced in four of six tubes No peptonization pH 5.3

TABLE 4 Differentiation of Streplumyces svit'ous and .S'rrepmmyces lmwiiensix ATCC l2236. UC 2504 Slreplomyr'et' .rvit'eus Slrepmmvces ltawaiienxis ATCC l2236. UC 2504 Reference color" Sporophores Spore surface' Gelatin (Plain and Nutrient) Litmus milk Tyrosine agar (lSP-7) Antibiotic production White (W) to Gray (GY) Long, straight and coiled at tip (S) (Many candelabralike) Warty to spiny (Spines short and sparse) N0 liquefaction 5071 Melanin positive & 5071 Melanin negative Produces AT- l 25 Complete liquefaction pH 6.l

Melanin negative Produces bryamycin 'Tresner. H. D.. and E. J. Backus, I962. System of color wheels for streptomycete taxononty. Appl. Microbial. ll1335-33ll.

'Pridham. T. G.. W. C. Hesseltine. and R. G. Benedict. 1958. A guide for the classification of Slrepmmyces according to selected groups. Placement of strains in morphological sections. Appl. Microbiol. 6:52-79. "Dieti. A.. and John Mathews. I970. Classification ofSrrepmmyt-r-x spore surfaces into five groups. Appl. Microbiol. 212527433.

NOTE: The "UC" designation in Table 4 refers to The Upjohn Company culture collection.

The new compound of the invention is produced when the elaborating organism is grown in an aqueous nutrient medium under submerged aerobic conditions. It is to be understood also that for the preparation of limited amounts, surface cultures and bottles can be employed. The organism is grown in a nutrient medium containing a carbon source, and an assimilable nitrogen compound or proteinaceous material. Preferred carbon sources include glucose, brown sugar, sucrose, glycerol, starch, cornstarch, lactose, dextrin, molasses, and the like. Preferred nitrogen sources include com steep liquor, yeast, autolyzed brewers yeast with milk solids, soybean meal, cottonseed meal, cornmeal, milk solids, pancreatic digest of casein, distillers solubles, animal peptone liquors, meat and bone scraps, and the like. Combinations of these carbon and nitrogen sources can be used advantageously. Trace metals, for example, zinc, magnesium, manganese, cobalt, iron and the like need not be added to the fermentation medium since tap water and unpurified ingredients are used as medium components.

Production of the compound of the invention can be effected at any temperature conducive to satisfactory growth of the microorganism, for example between about 18 and 40C., and preferably between about 20 and 32C. Ordinarily, optimum production of the compound is obtained in about 2 to ID days. The medium normally remains weakly acidic (pH 5.5-7.0) during the fermentation. The final pH is dependent, in part on the buffers present, if any, and in part on the initial pH of the culture medium which is advantageously adjusted to about pH 7.0 prior to sterilization.

When growth is carried out in large vessels and tanks, it is preferable to use the vegetative form, rather than the spore form, of the microorganism for inoculation to avoid a pronounced lag in the production of the new compound and the attendant inefficient utilization of the equipment. Accordingly, it is desirable to produce a vegetative inoculum in a nutrient broth culture by inoculating this broth culture with an aliquot from a soil or a slant culture. When a young, active vegetative inoculum has thus been secured, it is transferred aseptically to large vessels or tanks. The medium in which the vegetative inoculum is produced can be the same as, or different from, that utilized for the production of the new compound, as long as it is such that a good growth of the microorganism is obtained.

The new antibiotic of the invention, AT-l25, is an amphoteric compound. It is soluble in H 0 and slightly soluble in CH OH.

A variety of procedures can be employed in the isolation and purification of AT-l25, for example, absorption procedures followed by elution with a suitable solvent, column chromatography, partition chromatography, and crystallization from solvents.

In a preferred recovery process, AT-l is recovered from its culture medium by filtration through a medium porosity diatomite, for example FW 40 supplied by Eagle Picher. Other suitable diatomites are marketed under the trade names Super Cel (Johns Manvilles fine diatomite), Dicalite 4200 (Great Lakes), and Miraflo 40 (Eagle Picher).

The clear beer is percolated through a chromatographic column packed with a styrene type sulfonic acid resin. Dowex 50 in the hydrogen form is preferred, advantageously highly crosslinked, for example, Dowex 50 X 16. Other suitable resins are marketed under the tradenames Amberlite lR-l 20, Nalcite HCR, Chempro The column is washed with deionized water, 50%

aqueous MeOH, and aqueous MeOH, and then eluted with 0.1 N HOAc in 90% MeOH. Active eluate fractions are pooled, then evaporated to dryness under reduced pressureto give a residue. MeOH can be substituted by H O.

The residue, obtained as described above, is then subjected to partition chromatography by first dissolving it in the lower phase of a solvent system consisting of a mixture of n-butanol, benzene, methanol and deionized water (2:l:l:l). The lower phase containing the residue from the above-described weakly basic styrene type polyamine resin column is homogenized with a medium porosity diatomite, for example, Eagle Pichers FW 40 and upper phase of the solvent system described above. The resulting homogenized solid is then added to the top of a column containing a medium porosity diatomite which was prepared previously by slurring the medium porosity diatomite in upper phase of the solvent mixture, described above, agitated thor oughly with lower phase of said solvent mixture, pouring into a chromatographic column, and allowing the bed to settle while passing upper phase through the column. The active eluates from the column are collected, pooled and evaporated to a dry residue. Crystalline AT-l25 is obtained by crystallization with a suitable solvent (methanol is preferred). Other solvents which can be used are EtOH, aqueous BuOH, and H 0.

An alternate procedure for the recovery of AT-l25 from the filtered fermentation beer is by absorption on a suitable ion exchange resin, for example Dowex 1 (OAc) at pH 9-10, or IR 45 (OH') at pH 6-7, and afterwashing the resin with H O, the AT-l25 is recovered by elution with aqueous or methanolic NH,OH or HOAc. Further, the filtered clear fermentation beer may be absorbed on carbon and the AT-l25 activity eluted with an appropriate solvent mixture, for example, aqueous acetone.

An alternate purification procedure which can be used involves chromatography on silica gel with methanol in CHCL, or CH Cl or with mixtures of benzene (or toluene), 95% ETOH, and HOAc.

The titer of AT-l25 in the beer during various stages of recovery operations can be monitored by a discplate assay using Bacillus suhtilis cultivated in a synthetic medium of the following composition:

Na HPO,,7H-,O KH.,P0, NHmso, MgSO Glucose Bacto Agar* l Distilled water Metallic ion stock solution *Bacto Agar provided by Difco Laboratories. Detroit, Michigan. Metallic ion stock solution consists of the following:

NaMoO ,.2H O

*ZnCl has to be dissolved separately using a drop ot'0.l N HCl for l ml. of water. The stock solution is heated to bring all the compounds in solution, kept standing for 24 hours, and sterile filtered.

This medium is inoculated with a spore suspension of B. sublilis (1.5 X cells/ml.) at a rate of 0.5 ml./liter. The beer samples are applied to 12.5 mm. diameter adsorbent paper discs (0.08 ml./disc), the assay system is incubated overnight at 37 C., and the zones of inhibition are measured. The potency of the sample is related to the diameter of the inhibition zone by means of the usual standard curve.

This medium when seeded with B, subrilis can also be used for the detection of antibiotic AT-l25. In this procedure, papergrams are developed with the upper phase of a solvent mixture of l butanol, methanol, benzene, and H 0 (2:1:lzl). After development the sheet is dried and the papergrams are then laid on transparent papergram trays containing the seeded medium and withdrawn after about 20 minutes. The trays are incubated overnight as above, and inhibition zones observed.

Since AT-l25 is an amphoteric compound, it forms salts with acids, alkali metals (including ammonia), alkaline earth metals (including magnesium and aluminum), and amines. Metal salts can be prepared by dissolving AT-l25 in water, and adding a dilute metal base until the pH ofthe solution is 7 to 8. AT-l25 metal salts include the sodium, potassium and calcium salts. Amine salts. including those with organic bases such as primary, secondary, and tertiary, mono-. di-, and polyamines can also be formed using the above-described or other commonly employed procedures. Further, ammonium salts can be made, by well-known procedures. Other salts are obtained with therapeutically effective bases which impart additional therapeutic effects thereto. Such bases are, for example, the purine bases such as theophyllin, theobromin, caffein, or derivatives ofsuch purine bases; anithistaminic bases which are capable of forming salts with weak acids; pyridine compounds such as nicotinic acid amide, isonicotinic acid hydrazide, and the like; phenylalkylamines such as adrenalin, ephedrin, and the like; chloine, and others.

Acid salts can be made by neutralizing AT-l25 with the appropriate acid to below about pH 7.0, and advantageously to about pH 2 to pH 6. Suitable acids for this purpose include hydrochloric, sulfuric, phosphoric, sulfamic, hydrobromic, and the like. Acid and base salts of AT-l 25 can be used for the same biological purposes as the parent compound.

AT-l25 is active against Escherichia coli and can be used to reduce, arrest, and eradicate slime production in papermill systems caused by its antibacterial action against this microorganism. AT- 1 25 also can be used to prolong the life of cultures of Tric/mmonas foetus, Trichommzus hominis, and Triclmmonas vaginalis by freeing them of Escherichia coli contamination. Further. AT-l25 can be used as the antifungal agent in the shoe uppers as disclosed in U.S. Pat. No. 3,130,505. Still, further. since AT-l25 is active against Bacillus subtilis it can be used to minimize or prevent odor in fish or fish crates caused by this organism, or AT-l25 can be used to swab laboratory benches and equipment in a mycological laboratory.

AT-l25 is active against L 1210 murine leukemia in laboratory mice, and. thus. can be used to treat said mice.

The following examples are illustrative of the process and products of the present invention but are not to be construed as limiting. All percentages are by weight and all solvent mixture proportions are by volume unless otherwise noted.

EXAMPLE 1 A. FERMENTATION A soil stock of Streptomyces svcieus, NRRL 5439, is used to inoculate 500-ml. Erlenmeyer seed flasks containing ml. of sterile medium consisting of the following ingredients:

Dextrose 25 g./l. Pharmamedia 25 g./l. Tap water q.s. to l liter Product of Trader's Oil Mill Company. Fort Worth. Texas.

The seed medium presterilization pH is 7.2. The seed inoculum is grown for two days at 28C. on a Gump rotary shaker operating at 250 r.p.m. Seed inoculum, prepared as described above, is used to inoculate 500-ml. Erlenmeyer fermentation flasks containing 100 ml. of sterile fermentation medium consisting of the following ingredients:

Calcium carbonate 5 g./l. Sodium chloride 2 g./l. Starch l0 g./l. Mannitol l0 g./l. Phytone l0 g./l. KaySoy" l0 g./l.

Lard oil I mL/lOO ml. Tap water q.s. to 1 liter 'Papaic digest of soya meal. "Finely milled fat extracted soybean meal.

Hours Bu/ml. of AT-l 25 A biounit of activity (Bu) is defined as that quantity of antibiotic necessary to achieve a 20 mm. zone of inhibition from a one-half inch disc treated with 0.08 ml. of its solution. The disc-plate assay is as described previously.

B. RECOVERY Whole beer (250 liters) from an AT-l25 fermentation, as described above, is filtered through a medium porosity diatomite. The resulting clear beer is percolated at pH 7-8 through 25 liters of freshly regenerated Dowex 50 X 16 (H*) in a chromatographic column. The column is washed with 50 liters of deionized H 0 and then eluted with 120 liters of 1 N NH,OH; 6 liter fractions are collected. The most active fractions (inhibition zones 50 mm.) are determined by applying dipped, air-dried discs to a tray of Bacillus sublilis (grown in synthetic medium as described previously). The active fractions are pooled and concentrated under reduced pressure to remove excess NH OH. Solids determinations indicate that the crude aqueous concentrate contains 2950 g. and the active eluates contain 244 gm. The solids in the active eluates are approximately 13 times more potent than the clear beer solids by Bacillus subtilis synthetic assay.

C. PURIFICATION l. Weakly Basic Styrene Type Polyamine Resin Column The active aqueous concentrated Dowex 50 eluate, prepared as described above, at pH 7-7.5, is percolated through a column containing 4 liters of Amberlite IR 45 (OH). The column is washed with 8 liters of deionized H 0, 4 liters of 50% aqueous MeOH and 8 liters of 90% aqueous MeOH, and then eluted with 0.5 N HOAc in 90% MeOH; 2 l. fractions are collected. The most active fractions by Bacillus sublilis assay, are pooled and evaporated to dryness under reduced pressure; yield, 9.40 g. of residue having a Bacillus suhtilis potency 28 times greater than the Dowex O eluate residue, described above.

2. Partition Chromatography A mixture of n-butanol, benzene, methanol, and deionized water (2:1: 1:1 is agitated and allowed to settle into two layers; the layers are then separated. A medium porosity diatomite (2340gm.) is slurried in upper phase, agitated thoroughly with 900 ml. of lower phase, and poured into a chromatographic column, allowing the bed to settle while passing the upper phase through the column. The residue from the IR 45 eluate, prepared as described above, is dissolved in 25 ml. of lower phase and homogenized with 75 g. of a medium porosity diatomite and 50 ml. of upper phase. The resulting homogenized solid is carefully added to the top of the column and elution with upper phase initiated; 500 ml. fractions are collected. The most active fractions by Bacillus subiilis assay are evaporated to dryness under reduced pressure affording 3.93 gm. of residue having a Bacillus subtilis potency 2.9 times that of the IR 45 eluate residue, described above.

3. Crystallization The residue, obtained by partition chromatography, as described above, is crystallized from CH OH and recrystallized from aqueous CH OH three times affording 362 mg. of pure crystalline AT-l25 having approximately 4 times the Bacillus subtilis potency of the highly active residue from partition chromatography, described above.

Salts of antibiotic AT-l25 can be made by the procedures disclosed supra. These salts, also as disclosed supra, are useful for the same purposes as the parent antibiotic.

The invention described herein was made in the course of, or under, Contract PH43-NCl-69-l023 with the National Cancer Institute, National Institutes of Health, Bethesda, Md. 200l4.

We claim:

1. A compound of the formula:

and its salts.

2. Acid addition salts of antibiotic AT-l25, the compound of claim 1.

3. Cationic salts of antibiotic AT-l25, the compound of claim 1.

4. Zwitterion form of antibiotic AT-l25, the compound of claim 11.

5. Crystalline antibiotic AT-125, the compound of 

1. A COMPOUND OF THE FORMULA:
 2. Acid addition salts of antibiotic AT-125, the compound of claim
 1. 3. Cationic salts of antibiotic AT-125, the compound of claim
 1. 4. Zwitterion form of antibiotic AT-125, the compound of claim
 5. Crystalline antibiotic AT-125, the compound of the claim
 1. 